T-Cell Receptor Gamma Gene Rearrangements in the Investigation of Immunopathological Disorders

Karim, Sabeeha Naheed (1993) T-Cell Receptor Gamma Gene Rearrangements in the Investigation of Immunopathological Disorders. PhD thesis, University of Glasgow.

Abstract

Infiltration of the tissues by T-lymphocytes is a prominent feature of many human diseases including infections, autoimmune disorders, various poorly understood chronic inflammatory conditions, T-cell lymphomas and leukaemias and tumours of other tissues. Very little is known about the infiltrating T-cell clones in these conditions, apart from the T-cell lymphomas and leukaemias most of which are monoclonal, derived from a single transformed cell. The object of this thesis was to devise a sensitive method for the detection of T-cell clones in small tissue samples and to use the method in pilot studies on examples of human diseases whose lesions are infiltrated with T-cells. T-cell clones are distinguished from one another by their antigen receptors. Each clone arises in the thymus from a founder cell in which germline genes coding for antigen receptor molecules are altered (rearranged) in a particular way to give that cell an antigen-receptor of unique specificity, much clonal diversity being generated by the variety of possible rearrangements of the germline genes and deletions and random insertions of uncoded nucleotides at hypervariable (N) regions within these rearrangements. The polymerase chain reaction (PCR) has been adapted to amplify rearrangements of the human T-cell receptor gamma (TCFty) genes and high resolution polyacrylamide gel electrophoresis has been used to analyse the molecular size of the amplified products which include the hypervariable N regions. Since the N region in different clones varies in size by 40 or so nucleotides, in theory 1280 subsets of rearranged genes (clonotypes) can be distinguished by this method, if a total of 32 PCRs are performed to amplify most of the possible rearrangements. The method has been developed and validated with model systems employing DNA from cultured T-cell lines whose 7 gene rearrangements were already known from the literature. With appropriate primer combinations, PCR with as little as 1 nanogram of DNA from monoclonal T-cell lines gave a positive reaction, a dense dominant electrophoretic band of the expected molecular size and the dominant band could be demonstrated in the presence of 20-100 parts of polyclonal DNA from reactive (hyperplastic) lymph nodes. By itself polyclonal DNA produced electrophoretic smears (sometimes with some minor bands) which reflect the presence of multiple clones with a range of N region sizes. A battery of 8 PCRs for the most common TCR7 gene rearrangements successfully demonstrated dominant clonal rearrangements in 24 of 36 (67%) cases of malignant T-cell lymphoma in contrast to 1 of 12 cases of reactive lymph node hyperplasia, and none of 12 skin samples from 5 normal controls. Evidence was obtained suggesting that the dominant clonotype detected in plaques of cutaneous T-cell lymphoma is also present in smaller amounts in clinically unaffected skin but not in blood from the same patient. The latter finding and observations on blood and skin from normal subjects are consistent with the existence of a subset of T-cell clones which selectively home to the skin. The rest of the study investigated a recent modification of Burnet's hypothesis that forbidden clones of autonomously functioning neoplastic Tcells at the benign end of spectrum are the underlying cause of vitiligo, psoriasis and rheumatoid arthritis - poorly understood immunopathological disorders whose lesions are infiltrated by T-cells. A dominant clonal band was demonstrated in only 1 of 28 vitiligo lesions from 10 patients and in none of 29 psoriatic lesions from 13 patients. Possible examples of minor TCR7 gene rearrangements restricted to the lesions of vitiligo or psoriasis were found respectively in 1 and 3 of 4 patients with both diseases. Dominant bands possibly reflecting the presence of latent low grade T-cell neoplasia were detected in DNA from peripheral blood in 3 of 8 patients with vitiligo and 2 of 12 patients with psoriasis. It is unlikely that these are of pathogenic importance since similar dominant bands were also found in the blood in 2 of 5 normal control subjects. In rheumatoid arthritis dominant bands were detected in diseased joints in 4 of 20 cases. Direct evidence was obtained that in vitro culture in lnterleukin-2 (IL-2) has significant effects on the relative abundance of Tcell clones in samples of synovial fluid and that investigations based on analysis of cultured synovial lymphocytes are likely to give misleading results. The finding of dominant rearrangements in the lesions of only a minority of cases of vitiligo, psoriasis and rheumatoid arthritis does not support the hypothesis that T-cell neoplasia at the benign end of the spectrum is the underlying cause of any of these diseasesbut the possibility of involvement of small (non-dominant) benign neoplastic T-cell clones which selectively localise to the lesions in these conditions cannot be excluded.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: R B Goudie
• Keywords: Immunology, Pathology
• Date of Award: 1993
• Depositing User: Enlighten Team
• Unique ID: glathesis:1993-75606
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 19:19
• Last Modified: 19 Nov 2019 19:19
• URI: https://theses.gla.ac.uk/id/eprint/75606